The goal of this proposal is to address the role of the Id (inhibitor of differentiation) genes in specific cardiac compartments during development. Idl, 2 and 3 are expressed in the developing heart in the developing epicardium, endocardium and endocardial cushions (ECs), but not in the myocardium. Idl, Id2 or Id3 gene ablation studies revealed no developmental defects, but Idlld3, Idl Id2 and Id21d3 double mutant embryos die by E13.5. Ablation of Idl-3 in varying combinations resulted in cardiac defects not only in the endocardium and in the ECs, but also in the myocardium, suggesting cross-communication between layers. Id deficient embryos displayed impaired trabeculae and interventricular septum, thin myocardial wall, stenotic outflow tract, disorganized endocardium and hypocellular ECs. Injection of LacZ-marked Embryonic Stem (ES) cells reversed the cardiac abnormalities and rescued the embryonic lethality of the Idlld3 KO embryos. Altered myocardial markers in the Id KO embryos are restored to normal in the mutant cells of the rescued hearts. Thus, The ES-derived cells display the capacity to reverse gene expression profiles in mutant cells in a non-cell autonomous fashion. These observations raise important biological questions: What is the role of Id in the epicardium, endocardium/EC and in the myocardium? Is the Id mode of action non-cell autonomous? Which are the Id-dependent signals involved in the cross-talk between cardiac layers? Aiming to answer these questions we propose to study the role of Id genes in adult hearts of rescued chimeras (AIM I), to study the requirement for Id genes in specific cardiac compartments (AIM II), and to identify key mediators of Id signaling (AIM III). We will use murine models to extend the studies conducted with ES cells. Additionally, we will generate conditional KO and tissue-specific transgenic models. Some of the experiments will be complemented by tissue culture studies. The Sloan-Kettering Institute for Cancer Research provides the appropriate environment required to carry out this project, including the mouse transgenic, genomics core, and histology core facilities. The long term goals are to develop murine models to better understand the role of developmental control genes in cardiac formation, how mutations in those genes lead to congenital heart disease, and how these abnormalities could be prevented or ameliorated by embryonic stem cell injection. These studies may have important implications for congenital heart disease.